Retinoids are essential regulators of epithelial cell growth and cellular differentiation, skin being a major target in both normal and pathological states. Sporn, M. B. et al., Cancer Res. 43:3034-3040 (1983); Kopan, R. et al., J. Cell Biol. 109:295-307 (1989); Asselineau, D. et al., Dev. Biol. 133:322-335 (1989); and Lippman, S. M. et al., Pharmacol. Ther. 40:107-122 (1989). It has been shown that retinoids prevent cancer in skin and have efficacy as agents in human malignant and premalignant cutaneous disorders. Asselineau, D. et al., Dev. Biol. 133:322-335 (1989). It has also been shown that retinoids cause growth inhibition in many hyper-proliferating cell lines, a feature that makes the compounds of fundamental interest as anti-tumor and anti-psoriatic agents. Sporn, M. B. et al., Cancer Res. 43:3034-3040 (1983); and Asselineau, D. et al., Dev. Biol. 133:322-335 (1989). Retinoids also play fundamental roles in directing the spatial organization of cells during development and the generation of vertebrate limbs. Eichele, G. Trends Genet. 5:246-251 (1989); and Summerbell, D. et al., Trends Neurosci. 13:142-147 (1990).
The elucidation of the function of retinoids in the complex biological processes involved in cell growth and differentiation requires the identification of the specific components of the retinoid signal transduction system as well as the genes directly regulated by this system. Several intracellular retinoid-binding proteins have already been identified, including cellular retinol-binding proteins (CRBP), nuclear retinoic acid receptors (RAR), cellular retinoic acid binding proteins (CRABP) and, most recently, RXRs, also belonging to the nuclear receptor superfamily of genes. See Sundelin, J. et al., J. Biol. Chem. 260:6488-6493 (1985); Li, E. et al., PNAS (USA) 83:5779-5783 (1986); Nilsson, M. H. L. et al., Eur. J. Biochem. 173:45-51 (1988); Stoner, C. M. et al., Cancer Res. 49:1497-1504 (1989); Giguere, V. et al., PNAS (USA) 87:6233-6237 (1990); Petkovich, M. et al., Nature 330:444-450 (1987); Brand, N. et al., Nature 332:850-853 (1988); Benbrook, D. et al., Nature 333:669-672 (1988); Zelent, A. et al., Nature 339:714-717 (1989); Krust, A. et al., PNAS (USA) 86:5310-5314 (1989); and Mangelsdorf, D. J. et al., Nature 345:224-229 (1990).
Cellular retinoic acid binding proteins (CRABP) are low molecular weight proteins present in human skin with increased levels found in psoriatic lesions and after external and systemic retinoid treatment. Siegenthaler, G. et al., J. Invest Dermatol. 86:42-45 (1986); Hirschel-Scholz, S. et al., Eur. J. Clin. Invest. 19:220-227 (1989); and Siegenthaler, G. et al., Arch. Dermatol. 123:1690-1692 (1987). Although undetectable in keratinocytes grown in low calcium medium, CRABP is expressed when a more differentiated phenotype is induced by growth to confluence in the presence of elevated extracellular calcium concentrations. Siegenthaler, G. et al., Exp. Cell Res. 178:114-126 (1988).
Although the precise role of CRABP in retinoic acid (RA) action has not been determined, it has been suggested that CRABP might act as a shuttle protein, facilitating the movement of RA to the nucleus, or that CRABP might sequester RA, thereby decreasing the cellular response. Takase, S. et al., Arch. Biochem. Biophys. 247:328-334 (1986); Maden, M. et al., Nature 335:733-735 (1988). In a recent study, it was found that all biologically active RA analogs in F9 cells bound to RARs, while two of them did not bind to CRABP. Benbrook, D. et al., Nature 333:669-672 (1988). This suggests that retinoid binding to RA, but not necessarily to CRABP is necessary to induce cell differentiation.
The effects of retinoic acid on gene transcription can be mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Leed, M. et al., Cell 68:377-395 (1992); Mangelsdorf, D. J. et al., Genes Dev. 6:329-344 (1992). RARs have been shown to bind retinoic acid (RA) with high affinity, while RXRs apparently have no affinity for this ligand. Mangelsdorf, D. J. et al., Nature 345:224-229 (1990). However, it was recently demonstrated that 9-cis RA can bind to RXR-.alpha. with high affinity. Levin, A. A. et al., Nature 355: 359-361 (1992); Heyman, R. A. et al., Cell 68:397-406 (1992). CRABPs have been shown to bind RA with high affinity, but their function is poorly understood. However, it was recently demonstrated that CRABP may be involved in cytochrome P-450 metabolism of RA. Fiorella, P. D. et al., J. Biol. Chem. 266:16572-16579 (1991).
Two isoforms of CRABP, CRABP-I and II, have been identified and cloned in the mouse. Stoner, C. M. et al., Cancer Res. 49:1497-1504 (1989); Giguere, V. et al., PNAS (USA) 87:6233-6237 (1990); Nilsson, M. H. L. et al., Eur. J. Biochem. 173:45-51 (1988). By isoform is meant two amino acid sequences with substantial sequence identity. Bovine CRABP-I has also been sequenced, and the NH.sub.2 terminal regions of rat and chicken CRABP-I have also been recently determined. Bailey, J. S. et al., J. Biol. Chem. 263:9326-9332 (1988); Kitamoto, J. et al., Biochem. Biophys. Res. Comm. 157:1302-1308 (1988). Human CRABP has, however, not been previously isolated or cloned.